原位定量评价组织工程研究中细胞生长状态的新方法

A novel method to detect the viability of the cells quantitatively in situ on the scaffold

目的探讨一种能原位定量评定细胞在高分子聚合物载体上生长情况的方法。方法采用CFDA-AM和PI双染结合激光扫描共聚焦显微镜技术,用平均荧光强度代表细胞数量,对组织工程骺软骨中骨骺软骨细胞在聚乳酸载体上1、2及3周时的死活细胞数量进行评价。结果骨骺软骨细胞在聚乳酸载体上的530nm的平均荧光强度随时间逐渐增加,而603nm的荧光强度在第2周时有所增加,而到第3周时则无明显增加。与细胞在载体上重量增加的结果一致。结论首次采用的原位定量检测载体上死活细胞的方法是可行的,解决了在不透明载体上即不影响细胞和载体结构,而可以原位快速定量评定细胞生长的难题。

Objective To develop a method which can detect t he viability of the cells quantitati vely in situ on the polymer scaffold without destroying the shape and structure o f the engineered tissue.Methods The live and dead growth plate chondr ocytes on the PLA polymer were studied quantitatively by combinin g the technique of double fluorescen t staining ,in which CFDA-AM and PI we re selected as fluorescent probe,and that of Laser scanning con focal microscope.The fluorescence intensities of CFDA-AM and PI were used as the amount of live and dead cells respectively and were detected 1week,2weeks and 3weeks after cell seeding.Results The mean intensities of green fluore scence representing the numbers of live cells increased gradually and the mean i ntensities of red fluorescence repr esenting the numbers of dead cells stopped increasing at 2weeks after s eeding.Conclusions The novel method could detect the via bility of the cells quantitatively i n situ on the scaffold without distortion in scaffold architecture due to polymer dis solution and disruption during processing and could assess the cellular distribution within a biodegradabl e scaffold.